The discovery of the anti-viral APOBECS proteins is one of the most therapeutically promising breakthroughs in HIV/AIDS molecular virology in recent years. Humans have seven APOBECS proteins and at least two, APOBECSG and APOBECSF, are capable of inhibiting the replication of Vif-deficient HIV. However, HIV pathogenesis is due at least in part to the fact that the viral Vif protein counteracts these APOBECS proteins and triggers their degradation. The molecular approaches described in this proposal are an integral part of a larger program project to provide comprehensive knowledge of the structural, biophysical, biochemical and molecular features of these APOBECS proteins and their relation to HIV-1. First, we will test the hypothesis that APOBECSG dimerization occurs through multiple direct protein-protein interactions, and we will determine the relevance of these interactions to HIV restriction. Second, we will distinguish between two models for how APOBECSG binds single-strand DNA substrates analogous to HIV cDNA. Third, we will test the hypothesis that HIV Vif recognizes a common structural motif that is present in APOBECSF, APOBECSG, and other APOBECS proteins. We will use structural information from our program collaborations to guide the construction of APOBECS and Vif mutants. These studies will be aided in part by a panel of novel APOBECSG inhibitory small molecules that will be used as molecular probes to dissect these critical steps of the AP0BEC3G/F-mediated HIV-1 restriction mechanism. These studies will advance our fundamental understanding of APOBECSG and APOBECSF and facilitate the development of novel HIV/AIDS therapeutics that work by modulating the APOBECS-Vif pathway.